The molecular-level origins of disease are being elucidated at a rapid pace, potentially ushering in a new era of personalized medicine in which a specific course of therapy is developed for each patient. To fully exploit this expanding knowledge of disease phenotype, new methods for detecting multiple biomolecules (e.g., DNA and proteins) simultaneously are required. The multiplex biomolecule detection methods must be rapid, sensitive, highly parallel, and ideally capable of diagnosing cellular phenotype in vivo.
Some biomolecule detection methods have been developed based upon the unique electrochemical and photoelectrochemical properties of metal particles. In one assay method, gold nanoparticles (10 nm diameter) are tagged with ssDNA probe strands and a photoactive dye molecule. A metal electrode of a microarray chip (also called gene chip) is also modified with ssDNA probe strands. If a target (the analyte or bioanalyte) mRNA or ssDNA is complementary to the probe on the particle and the substrate, hybridization will occur which brings the particle in contact with the electrode. A laser is then rastered across the surface. When the laser addresses a spot in which nanoparticles are bound, the dye molecule is electronically excited, and the excited electron is injected into the electrode. The electron is collected as a current, signifying the presence of a particular DNA analyte.
Synthesis of a functionalized electrode having polymer arrays on an electrode of a microarray chip is known. Examples of such polymer arrays include nucleic acid arrays, peptide arrays, and carbohydrate arrays.
One method of preparing functionalized electrodes of polymer arrays on microarray chips involves photolithographic techniques using photocleavable protecting groups. Briefly, the method includes attaching photoreactive groups to the surface of a substrate, exposing selected regions of the substrate to light to activate those regions, attaching a monomer with a photoremovable group to the activated regions, and repeating the steps of activation and attachment until macromolecules of a length and sequence are synthesized.
Additional methods and techniques applicable to prepare a functionalized electrode include electrochemical synthesis. One example includes providing a porous substrate with an electrode therein, placing a molecule having a protected chemical group in proximity of the porous substrate, placing a buffering solution in contact with the electrode and the porous substrate to prevent electrochemically generated reagents from leaving the locality of the electrode (the use of confinement electrodes to prevent reagents from diffusing away have also been described), applying a potential to the electrode to generate electrochemical reagents capable of deprotecting the protected chemical functional group of the molecule, attaching the deprotected chemical functional group to the porous substrate or a molecule on the substrate, and repeating the above steps until polymers of a length and sequence are synthesized.
The biomolecules on microarray chip typically are detected through optical readout of fluorescent labels attached to a target molecule that is specifically attached or hybridized to a probe molecule. These optical methods are difficult to implement and miniaturize because they rely on the use of optical labels and require large or expensive instrumentation.